The invention relates to a method and a device for controlling the number of ions in ion cyclotron resonance (ICR) mass spectrometers, whereby the ions enter a multipole ion guide after their formation and are stored there temporarily. By measuring the ion number in a predefined subset of these temporarily stored ions, the number of ions transferred into the ICR trap for mass spectrometric analysis is regulated.
Conventional methods of ionizing the substances for mass spectrometric analysis such as electron impact, cannot be applied to large organic or biomolecules. These species can neither be transferred into the gas phase by thermal energy supply without being decomposed, nor can they be ionized by electron impact without being fragmented. Contemporary mass spectrometry very frequently uses electrospray or matrix assisted laser desorption ionization (MALDI), which offer much milder ionization conditions to the large molecules.
Electrospray ionization is probably the most frequently used ionization method for the large molecules. A review article about the mechanism of the electrospray ionization was published by P. Kebarle and L. Tang in xe2x80x9cAnalytical Chemistryxe2x80x9d 65, 972A-986A (1993). Using this method ions are generated at atmospheric pressure under the influence of high voltage (3-6 kV) between an electrospray needle and a counter electrode. Although the spray process is often supported by a slow and fine adjustable syringe pump, the separation of the small charged droplets as a result of the high ion density on the liquid surface (Coulomb repulsion) is the primary driving force of the spray process. A xe2x80x9cdrying gas that flows in counter current to the flight of the charged droplets leads to the evaporation of the solvent (desolvation process) and thus to the reduction of the droplet radii. Due to the increasing Colulomb repulsion forces the ionized molecules are evaporated and often multiply protonated. These ions are transferred through a capillary through a multi-stage vacuum system and through a multipole ion guide into the mass spectrometer for measurement. Electrospray ionization at atmospheric pressure has dramatically simplified linking separation methods, such as liquid chromatography or capillary electrophoresis, directly to the mass spectrometry.
Laser desorption ionization (LDI) has long been used to successfully transfer large organic molecules into the gas phase and to ionize them. A special kind of LDI is the matrix assisted laser desorption ionization (MALDI). The review article by E. J. Zaluzec, D. A. Gage, J. T. Watson in Protein Expression and Purification 6,109-123 (1995) reports about MALDI applications for characterization of proteins and peptides. A MALDI paper by H. J. Rxc3xa4der and W. Schrepp about the analysis of synthetic polymers with the aid of time of flight mass spectrometry can be found in Acta Polymer. 49, 272-293 (1998).
In MALDI the analyte molecules are mixed with a so-called matrix. The molar ratio of the matrix to the analyte is usually 1:102 to 1:104. The energy of the laser beam is absorbed by the matrix molecules and transferred to the analyte molecules. The latter thus obtain the necessary energy to transition in the gas phase and become thereby partially ionized. The ionization mostly happens in form of a proton acceptance. Compounds that are used as matrix are mostly proton donors. In special cases, alkaline metal salts or silver salts can be used as additives in order to achieve a corresponding metal ion attachment.
In classical cases of MALDI time of flight mass spectrometry, ions are extracted out of the source region using a high voltage pulse and accelerated into the flight tube. Contrary to the MALDI time of flight mass spectrometry, in high RF ion traps (Paul traps) and electromagnetic ion traps (Penning traps, ion cyclotron resonance and Fourier transform ion cyclotron resonance mass spectrometry) one wants to generate low-energy ions, in order to be able to capture them in the corresponding ion trap without sustaining any losses. Consequently, ions are not accelerated to energies of several kilo electron volts.
In the low energy extraction of MALDI generated ions, the variation of excessive energy gets more evident and causes difficulties even during capturing these ions. It leads to a considerable fluctuation of the generated mass signals and therefore to irreproducible analytical results. A low voltage MALDI ion source is described by A. N. Krutchinsky, A. V. Loboda, V. L. Spicer, R. Dworschak, W. Ens, K. G. Standing in xe2x80x9cRapid Communications in Mass Spectrometryxe2x80x9d 12, 508-518 (1998), where the ions are desorbed directly into a quadrupole. Since the ions are practically formed in the quadrupole they are efficiently captured. However, the ions are here not collected and trapped in the quadrupole. The quadrupole is here used solely as an ion guide in order to transfer ions into the time of flight mass spectrometer.
One of the important differences between ion trap mass spectrometry and ion transmission mass spectrometry is caused by the limited ion storage capacity of the ion traps. Overloading an ion trap is as undesirable as having insufficient number of ions. Methods for controlling the number of ions in RF traps are described in the patents U.S. Pat. No. 5,107,109, DE 43 26 549. These patents describe a controlled generation of ions by electron impact in the trap by regulating the ionization time of the analyte molecules. In the first case the number of ions is determined by a pre-measurement of the ion charge in the trap and regulated in the immediately following measurement. In the second patent, the actual value of the number of ions is extrapolated from integration of several of the preceding mass spectrometric measurements and used for the control. The patent U.S. Pat. No. 5,739,530 also describes a controlled ion filling from a multipole ion guide system to quadrupole ion traps.
The pulsed generation of ions by MALDI or LDI shows fundamental differences from ion formation in a continuous-operating ion source. In this case, the ionization is triggered by individual laser pulses, which transfer the small molecules in a crystalline (or liquid) matrix into the gas phase and ionize them in part. During every laser pulse the surface of the sample is also modified and rearranged, small cradles formed, while part of the matter is eroded from the surface. As a result the xe2x80x9cion picturexe2x80x9d of the next laser pulse is not necessarily a reproduction of the one from the preceding pulse. That is, the number of ions transferred into the gas phase, as well as the intensity ratio of analyte ions to the matrix ions can vary significantly from laser pulse to laser pulse. Consequently, a varying space charge is caused in the trap.
The determination of the ion mass in the FTICR trap is performed by a frequency measurement. Due to the space charge in the trap, this frequency shifts. Therefore, a xe2x80x9creduced cyclotron frequencyxe2x80x9d is measured, which depends on the strength of the space charge. The publication of J. B. Jeffries, S. E. Barlow and G. H. Dunn, International Journal of Mass Spectrometry and Ion Processes 54, 169-187, (1983) describes these space charge mass shift effects theoretically. If the number of ions varies from scan to scan and are not regulated, this can cause each time a corresponding shift in the mass signal.
At high ion densities, another undesirable phenomenon appears, the so called xe2x80x9cpeak coalescencexe2x80x9d. Signals of ions with a very small mass difference, approach to each other and finally coalesce. The product of this coalescence is usually another sharp peak. In MALDIFTICR mass spectrometry peak coalescence phenomena are frequently observed due to uncontrolled number of ions which are transferred to the ICR trap.
If several scans have to be added up in order to increase the signal-to-noise ratio, these frequency shifts lead to problems. The varying number of ions of two consecutive ionization processes (e.g. MALDI) produces a varying space charge and varying mass shifts in each acquired spectrum. When adding up spectra, this effect presents itself as a peak broadening and thus leads to a loss of the mass resolution. Nowadays, the FTICR mass spectrometry is used largely because of its high mass accuracy and mass resolution. Therefore, even very small mass shifts caused by space charge effects mean a substantial loss in performance.
Although the difficulties with the variation of the mass spectrometric signal show up particularly during the ionization method MALDI, it is also observed during other ionization methods. The electrospray ionization also shows considerable fluctuations of ion formation. In addition, the electrospray technique allows coupling with chromatographic or electrophoretic separation methods, which in turn cause very strong time dependent concentration changes (in e.g. chromatographic peaks) and have also to be balanced out.
For this reasons it is evident that a control of the space charge in ICR traps is extremely important.
It is the objective of the invention to develop a method and a device in order to avoid in ion trap mass spectrometers substantial ion number variations, which are caused by fluctuating generation of ions and the associated space charge effects.
The invention consists of capturing and storing the ions after their formation initially in a multipole ion guide and to store them there temporarily, measuring a predefined subset of these ions and subsequently, using the result of this measurement, controlling the ion cyclotron resonance trap filling process.
For temporarily storing the ions in the multipole ion guide system, it is necessary to apply reflection potentials to the beginning and to the end of the multipole ion guide system. This can be performed using apertured plates, while skimmers can also be considered as apertured plates. At the entrance end, the MALDI sample carrier plate can provide the potential for trapping the ions. The potential at the exit end must be switchable in order to be able to switch from storage mode to the extraction mode.
The filling of the ion trap from the ion guide depends on the number of ions in the ion guide, the so-called level of filling. In general, the filling rate depends on the level of filling. The storing multipole ion guide can consist of several individual multipole systems.
In the case of the ionization by MALDI, the temporary storage of the ions in the multipole ion guide has the additional advantage, that ions produced by several individual laser shots can be accumulated in the multipole guide and then transferred into the ion trap. On one hand, at insufficiently small analyte concentrations, the analyte ions can be enriched by using several laser shots. On the other hand, if there are too many ions produced, this can be established by the preliminary measurement, and considered during the ion trap filling process, in order to keep the space charge under control.
Another advantage of this method is that ions in the multipole ion guide lose their excess kinetic energy within a short time period by collisions with gas molecules if the multipole ion guide is in a section of the mass spectrometer with a slightly elevated pressure. As a result, the probability of capturing the ions in the ion trap mass spectrometer improves.
Furthermore, with the aid of the multipole ion guide undesirable ions can be filtered out before they are transferred to the ion trap for mass analysis. The presence of undesirable ions means nothing but additional space charge. Filtering ions in a multipole with rod-shaped electrodes is a well known process, each multipole has a low mass cutoff limit which only depends on electrical and mechanical parameters of the multipole.